Germline stem cells (GSCs) are critical for reproduction, retaining the capacity to differentiate into gametes and pass genetic information on to the next generation. GSCs depend on both intrinsic and extrinsic signals to home to their niche, maintain potency, and begin meiotic division to differentiate into gametes. Botryllus schlosseri is a marine chordate with several characteristics that make it a useful and attractive model for elucidating the mechanisms of GSC biology. (1) First, adult Botryllus can cycle through phases of fertility and infertility, while continuously maintaining a population of GSCs. By comparing the whole transcriptomes of fertile and infertile Botryllus adults, we have identified a Tgf ligand, Tgf-f, upregulated in fertile animals that is expressed by follicle and follicle-progenitor cells both inside and outside of the gonadal niche. Tgf-f is also simultaneously upregulated in oocyte follicle cells and downregulated in testicular follicle cells during maturatin of the gonads. I hypothesize that Tgf-f and Tgf signaling may play a role in the maintenance, homing, and differentiation of Botryllus GSCs, and I will test this hypothesis in Aim 1 of this proposal by examining these processes in Tgf loss of function animals. (2) Second, Botryllus exhibits a phenomenon called germ cell parasitism (gcp) in which a colony may fuse to a neighbor and replace its germline by the transfer of GSCs, and this can be recapitulated by experimental transplantation of a FACS-enriched stem cell population. Colonies that successfully replace the germline of their fusion partner are referred to as winners while those whose germlines are replaced are referred to as losers. I hypothesize that the phenomenon of gcp is produced by differences between individuals in the ability of their GSCs to proliferate and home to the germ cell niche. I further hypothesize that these differences are present at the level of gene expression or function within GSCs. I will test this hypothesis in Aim 2 of this proposal by FACS-isolating GSCs from gcp winners and losers and comparing their transcriptomes to identify genes and pathways associated with gcp success or failure. Dysregulation of GSCs can severly impact human health by leading to germ cell tumors or infertility. The broad questions being addressed in this proposed study are what mechanisms exist to ensure that GSCs home effectively to their target niche and once there, how they are maintained in an undifferentiated state in the adult organism. Answering these questions will lay the foundation for therapies in reproductive and regenerative medicine.